Mentions:
Next, we questioned whether pharmacological AMPK activation similarly enhanced PPARδ target gene mRNA expression as AMPK overexpression. In these experiments we used the allosteric AMPK activator A-769662. Analysis of the phosphorylation status of AMPK, its substrate ACC and ribosomal protein S6, which served as readout of mTOR (mechanistic target of rapamycin) activity, revealed that significant ACC phosphorylation was already observed at 50 μM A-769662, and continued to increase up to 500 μM A-769662 (Fig 2A). In contrast, significant down-regulation of phospho-S6 and up-regulation of phospho-AMPK was achieved only at 250 μM and 500 μM A-769662, respectively. Therefore, we used 500 μM of the drug in subsequent experiments. Measuring intracellular ATP did not show changes after exposure to these concentrations of A-769662, ruling out a loss of viability (data not shown). As shown in Fig 2B, treatment of primary human macrophages with A-769662 induced mRNA expression of the PPARδ target genes PDK4, CPT1a, and PLIN2 to a similar extent as the exposure of cells to the PPARδ ligand GW501516. Importantly, A-769662 significantly augmented PPARδ target gene expression in GW501516-treated cells. Similar results were obtained when analyzing mRNA expression of PPARδ target genes following macrophage treatment with GW501516 and salicylate, which has recently been recognized as another direct allosteric AMPK activator [37] (S1 Fig). Analysis of PPARδ target gene expression also revealed cell type-specific differences. Whereas the well-known PPARδ target gene angiopoietin-like 4 (Angptl4), which is a lipoprotein lipase inhibitor, was robustly induced in the THP-1 macrophage cell line, it was not induced in primary macrophages (S2 Fig).

Mentions:
Next, we questioned whether pharmacological AMPK activation similarly enhanced PPARδ target gene mRNA expression as AMPK overexpression. In these experiments we used the allosteric AMPK activator A-769662. Analysis of the phosphorylation status of AMPK, its substrate ACC and ribosomal protein S6, which served as readout of mTOR (mechanistic target of rapamycin) activity, revealed that significant ACC phosphorylation was already observed at 50 μM A-769662, and continued to increase up to 500 μM A-769662 (Fig 2A). In contrast, significant down-regulation of phospho-S6 and up-regulation of phospho-AMPK was achieved only at 250 μM and 500 μM A-769662, respectively. Therefore, we used 500 μM of the drug in subsequent experiments. Measuring intracellular ATP did not show changes after exposure to these concentrations of A-769662, ruling out a loss of viability (data not shown). As shown in Fig 2B, treatment of primary human macrophages with A-769662 induced mRNA expression of the PPARδ target genes PDK4, CPT1a, and PLIN2 to a similar extent as the exposure of cells to the PPARδ ligand GW501516. Importantly, A-769662 significantly augmented PPARδ target gene expression in GW501516-treated cells. Similar results were obtained when analyzing mRNA expression of PPARδ target genes following macrophage treatment with GW501516 and salicylate, which has recently been recognized as another direct allosteric AMPK activator [37] (S1 Fig). Analysis of PPARδ target gene expression also revealed cell type-specific differences. Whereas the well-known PPARδ target gene angiopoietin-like 4 (Angptl4), which is a lipoprotein lipase inhibitor, was robustly induced in the THP-1 macrophage cell line, it was not induced in primary macrophages (S2 Fig).